Method for preparation of artificial blood vessel using tube-type porous biodegradable scaffold having a double-layered structure and stem cell, and artificial blood vessel made by the same

Abstract

The present invention relates to a method for preparation of an artificial blood vessel using a tube-type porous biodegradable scaffold having a double layered structure and a stem cell, and an artificial blood vessel made by the same. Specifically, the present invention relates to a method for preparation of an artificial blood vessel by separately seeding a stem cell onto the inner membrane and an outer membrane of a tube-type porous biodegradable scaffold having a double layered structure, wherein the inner membrane and the outer membrane having different biodegradable polymer nano-fiber arrangements are continuously linked, and by inducing differentiation; and an artificial blood vessel made by the same.

What is claimed is: 
     
       1. A method of preparing an artificial blood vessel comprising:
 a) preparing a tube-type porous biodegradable scaffold having a triple layered structure, wherein the tube-type porous biodegradable scaffold having a triple layered structure comprises an inner layer wherein biodegradable polymer nano-fibers are randomly arranged, an intermediate layer wherein biodegradable polymer nano-fibers are randomly arranged, and an outer layer wherein biodegradable polymer nano-fibers are circumferentially arranged, and the tube-type porous biodegradable scaffold having a triple layered structure is prepared by,
 (i) forming the inner layer by electrospinning a solution containing polyethylene oxide as a biodegradable polymer to a mandrel rotating at a speed of 0.2-0.3 m/s; 
 (ii) forming the intermediate layer by electrospinning a solution containing poly(L-lactide-co-ε-caprolactone) as a biodegradable polymer to the mandrel rotating at a speed of 0.2-0.3 m/s; and 
 (iii) forming the outer layer by electrospinning a solution containing poly(L-lactide-co-ε-caprolactone) as a biodegradable polymer to the mandrel rotating at a speed of 3-4 m/s; 
 
 b) seeding mesenchymal stem cells onto the inner layer and outer layer, respectively, of the tube-type porous biodegradable scaffold having a triple layered structure; 
 c) applying mechanical stimulus to the stem cell-seeded tube-type porous biodegradable scaffold having a triple layered structure by:
 (1) applying a sheer stress to the mesenchymal stem cell-seeded tube-type porous biodegradable scaffold having a triple layered structure such that differentiation of the mesenchymal stem cells occurs; and 
 (2) applying a 3-5% strain to the mesenchymal stem-cell seeded tube-type porous biodegradable scaffold length having a triple layered structure, wherein the step of applying a tension force comprises a first tension force and a second tension force and the second tension force is greater than the first tension force; and 
 
 d) forming the artificial blood vessel which comprises vascular endothelial cells differentiated from mesenchymal stem cells at the inner layer surface and smooth muscle cells differentiated from mesenchymal stem cells at the outer layer surface. 
 
     
     
       2. The method of  claim 1 , wherein the step of seeding the mesenchymal stem cells onto the inner layer and outer layer, respectively, of the tube-type porous biodegradable scaffold having a triple layered structure comprises the steps of:
 a) suspending mesenchymal stem cells into an endothelial cell culture solution, and seeding the cell culture solution containing the mesenchymal stem cell onto the inner layer of the tube-type porous biodegradable scaffold having a triple layered structure; 
 b) fixing the mesenchymal stem cells to the inner layer of the tube-type porous biodegradable scaffold having a triple layered structure by rotating the tube-type porous biodegradable scaffold having a triple layered structure of step a) at the speed of 0.5 to 5 rpm for 1 to 6 hours; 
 c) suspending the mesenchymal stem cells into a smooth muscle culture solution, and seeding the culture solution containing the mesenchymal stem cells onto the outer layer of the tube-type porous biodegradable scaffold having a triple layered structure of step b); and 
 d) fixing the mesenchymal stem cells to the outer layer of the tube-type porous biodegradable scaffold having a triple layered structure by rotating the tube-type porous biodegradable scaffold having a triple layered structure of step c) at the speed of 0.5 to 5 rpm for 15 to 30 hours. 
 
     
     
       3. The method of  claim 1 , wherein, in the step of applying shear stress, the mesenchymal stem cell-seeded tube-type porous biodegradable scaffold having a triple layered structure is immersed into the endothelial cell culture solution, and the shear stress of 2 dyne/cm 2  to 5 dyne/cm 2  per unit area of the mesenchymal stem cell-seeded tube-type porous biodegradable scaffold having a triple layered structure for 20 to 30 hours by flow of the endothelial cell culture solution. 
     
     
       4. An artificial blood vessel made by a method according to  claim 1 . 
     
     
       5. The artificial blood vessel of  claim 4 , whose diameter is 2 mm to 5 mm. 
     
     
       6. The artificial blood vessel of  claim 4 , which comprises vascular endothelial cells differentiated from mesenchymal stem cells at the inner layer, and smooth muscle cells differentiated from mesenchymal stem cells at the outer layer.